The limiting effect of genome size on xylem vessel diameter is shifted by environmental pressures in seed plants

Abstract Current and previous studies have extensively studied the physiological and ecological consequences of genome size (GS) on plants because of the limiting effect of GS on cell size. However, it is still obscure whether such limiting effect could be shifted by environmental pressures, or not. Here, we compiled a global dataset comprised of GS, xylem vessel diameter (V dia), xylem hydraulic conductivity (K S), P 50 (xylem water potential at the loss of 50% maximum K S), and climate factors of 251 phylogeny and habitat divergent species from 59 families. The results showed that GS could limit the V dia of the species from the same family sampled in the similar climate conditions. But the expected positive relationship between GS and V dia became uncertain and even negative across different environmental conditions. V dia was strongly positively coordinated with mean annual temperature (MAT), mean annual precipitation (MAP), and potential evapotranspiration (PET). Furthermore, V dia as the anatomic foundation of plant hydraulic performance was strongly positively coordinated with K S and negatively coordinated with −P 50. The strong environmental selection on K S and P 50 explained the concerted regulation of V dia by environmental factors. The findings revealed the combined regulation of GS and environmental pressures on xylem cell size and thus affected plant eco‐physiological performance. The shifted cell size limiting effect of GS by environmental factors manifests plants great plasticity under changed environmental conditions.


| INTRODUCTION
Genome size (GS) is a key plant trait shaping plant physiological performance and ecology strategy (Pandit et al., 2014). The main perspective proposed that GS is positively coordinated with cell size (Commoner, 1964;Price et al., 1973), thus influencing the plant functional traits such as seed size (Knight et al., 2005;Thompson, 1990) and guard cell size (Beaulieu et al., 2008). Because of the cell size limiting effect by GS, the ecological and physiological consequences of GS have attracted great attention and were thought as the indicator of plant ecological strategy. For instance, genome downsizing has been proposed as the prerequisite for the success of angiosperms because small GS allows for easier construction of dense minute stomata cells and promotes efficient gas exchange compared with gymnosperms (Simonin & Roddy, 2018). In addition, species with relatively small GS are thought as the advantage of species invasiveness because of the invasive species with small GS with fast growth rate and resource acquisition rate compared with native species with large GS (Pandit et al., 2014;te Beest et al., 2012). Similar to angiosperm genome downsizing, the evolution of advanced vascular system has also been considered an indicator of plant success through the deep time (Brodribb, Carriqui, et al., 2020).
Among the vascular and hydraulic traits, K S and P 50 are the two dominant traits shaping species ecological performance, survival, and drought resistance across different climates and lineages (McDowell et al., 2019;Nardini & Luglio, 2014;Scoffoni et al., 2016). In specific, K S is quantified by the magnitude of xylem-specific hydraulic conductivity. Great K S is achieved by great vessel diameter (V dia ) because the water transport capacity scales with vessel diameter to the fourth power (Ooeda et al., 2017). Under water deficit conditions, the failure of the hydraulic transport system could directly result in plant mortality (Brodribb, Powers, et al., 2020). The index used to evaluate plant vulnerability to hydraulic failure is called hydraulic safety and is quantified by P 50 ; the water potential at half of the maximum hydraulic conductivity is lost, and the more negative value represents, the greater hydraulic resistance to water deficit. Typically, great V dia coordinated with less ÀP 50 (low safety) because of the high risk to embolism of wider vessels, vice versa (He et al., 2020;Liu et al., 2019;Santiago et al., 2018).
According to the GS limiting cell size theory, V dia should be limited by GS as well as the other cell types, that is, small GS is expected to be coordinated with small V dia and large GS coordinated with big V dia (Figure 1a). Besides the limiting effect of GS on V dia , environmental pressures are also the strong regulators on V dia . For environmental factors, precipitation and temperature are the key environmental pressures for K S and P 50 . In the region with low precipitation and transpiration demand, low K S and high ÀP 50 (low hydraulic efficiency and high hydraulic safety) are expected for species to acclimate to the environments with insufficient water supply and high embolism risk.
Because V dia is the fundamental anatomic structure of K S and P 50 , thereby, small V dia should be evolved in such environments. In contrast, in the moist and warm regions, species would evolve great K S and low ÀP 50 (high hydraulic efficiency and low hydraulic safety) to cope with adequate water availability and high transpiration demand, which is corresponding to great V dia (He et al., 2020). Combining the regulating roles of GS and environmental factors on V dia , we hypothesized that the species with small GS habituated in the environment would favor low K S and high ÀP 50 , because of species with both small GS and small V dia ; for the species with small GS habituated in the moist environments favored great K S and low ÀP 50 , the limiting effect of GS on V dia would possibly be shifted by environmental pressures (Figure 1b).
To test the hypothesis that the limiting effect of GS on V dia might be shifted by environmental pressures, we compiled a global dataset of 251 divergent seed plants comprised by GS, V dia , K S , mean annual temperature (MAT), mean annual precipitation (MAP), and potential evapotranspiration (PET). First, to prove the limiting effect of GS on V dia in the similar climate conditions, we analyzed the relationship between GS and V dia of the species sampled from the same sites in six families including both angiosperms and gymnosperms. Second, to explore whether such limiting effect of GS on V dia could be shifted by environmental pressures, we analyzed the relationship between GS and V dia of the species sampled across different sampling sites. Finally, we analyzed the relationships between environmental factors and V dia by analyzing the relationships of V dia with V dia , K S , and environmental factors. We hypothesized that the limiting effect of GS on V dia exists in the species distributed in the similar environmental conditions and the relationship between GS and V dia could be shifted across different environmental conditions; the fine regulation of environmental pressures on V dia could be explained by the environmental selection on K S and P 50 .

| Data compilation
We compiled a global dataset comprised of 251 species of globally distributed seed plants from 59 families (Table S1). The compiled dataset includes GS, xylem vessel diameter (V dia ), xylem hydraulic conductivity (K S ), P 50 (xylem water potential at the loss of 50% maximum K S ), MAT, MAP, and PET. GS information of all studied species was F I G U R E 1 The conceptual model of genome size and environmental factors regulate xylem vessel size. (a) According to the genome size limiting cell volume, small genome size coordinated with small vessel size, vice versa. (b) The relationship between genome size and vessel volume becomes uncertain under the changed climate conditions. In such condition, the environmental factors would shift the limiting effect of genome size on vessel size. extracted from the Plant DNA C-values database (https://cvalues. science.kew.org/) (Pellicer & Leitch, 2020), which is an open-accessed genome dataset constructed by Royal Botanic Gardens, Kew. In order to assure the data matching of the dataset, we directly extracted V dia , K S , P 50 , MAT, MAP, and PET from a high-quality dataset recently published by Liu et al. (2019). In the newly compiled dataset, GS, traits data, and environmental factors were well matched.

| Data analysis
We first analyzed whether the limiting effect of GS on V dia could be shifted by environmental pressures. Because of the great GS differences of the species from different families, we analyzed the relationships between GS and V dia of the species from the same family in the similar and across different environmental conditions. To exclude the environmental effects on the relationship between GS and V dia , we analyzed the relationship between GS and V dia of the species of six families representing angiosperms and gymnosperms sampled at the same sampling sites. To illustrate whether environmental pressures could shift the limiting effect of GS on V dia , we analyzed the relationships between GS and V dia across different sampling sites. Furthermore, to further prove the uncertain relationships of GS and V dia across different environmental conditions, the overall relationships between GS and V dia of another species from eight families were analyzed. Simple linear model function lm in R programming was used to get the overall change trends of V dia along GS of the species from different families in the same and across sites.
Second, we analyzed how the environmental pressures regulating V dia . The relationships of V dia with MAT, MAP, and PET were analyzed for both angiosperms and gymnosperms. Because V dia is the fundamental anatomic foundation of K S and P 50 , we further analyzed the relationships of V dia with K S and P 50 . The coordination relationships between environmental factors and K S and P 50 were analyzed as well.
To homogenize variance, all the continuous variables were naturally log-transformed. The coordination among variables was analyzed by standardized major axis analysis (SMA) by smatr package in R (Warton et al., 2012). and V dia were shown as well ( Figure S2). The positive relationships between GS and V dia were only found in three families ( Figure S2b,d, f), and the negative relationships were found in another five families ( Figure S2a,c,e,g,h).

| V dia was finely tuned by environmental factors attributed to the environmental selection on hydraulic performance (K S and P 50 )
The results showed that V dia was positively coordinated with MAT, MAP, and PET ( Figure S3). The similar relationships between V dia and environmental factors were both significant in angiosperms and gymnosperms (Figure 4a-d). The results showed that V dia was negatively coordinated with ÀP 50 and positively coordinated with K S (Figure 5).
The relationships of environmental factors with K S and P 50 showed that ÀP 50 was negatively coordinated with MAT, MAP, and PET; on the contrary, K S was coordinated with MAT, MAP, and PET, which implies that environmental pressures showed strong selecting power on hydraulic performance (hydraulic efficiency and safety). The relationship of ÀP 50 with K S revealed that the trade-off was obviously between hydraulic efficiency and safety ( Figure S4).

| DISCUSSION
In the present study, we found that the limiting effect of GS on V dia could be shifted by environmental pressures. Under the similar environmental conditions, the positive relationship was found between GS and V dia . Once the environmental conditions changed, the relationships between GS and V dia became uncertain, in which, the relationships still keep positive or even changed negatively. V dia as the fundamental anatomic structure of K S and P 50 was strongly  Table S2.
F I G U R E 5 The relationships of vessel diameter with ÀP 50 and K S . (a) V dia is negatively coordinated with ÀP 50 in angiosperms and gymnosperms (n = 159). (b) V dia is positively coordinated with K S in angiosperms and gymnosperms (n = 132). V dia , vessel diameter; K S , xylem hydraulic conductivity; ÀP 50 , xylem water potential at the loss of 50% maximum K S . Green and yellow circles represent angiosperms and gymnosperms, respectively. Red lines represent the curved line that pooled all angiosperms and gymnosperms points. All the curved lines are at a significant level (P < .05). The model parameters are reported in Table S2. cell size limitation by GS implies the strong plasticity of seed plants under the changed climates.
According to the widely accepted GS eco-physiological consequences that GS could limit cell size (Beaulieu et al., 2008;Commoner, 1964;von Sachs, 1893), the similar limiting effect of GS on vessel cell size should also be observed. As expected, we indeed found that GS was positively coordinated with V dia of the species sampled from same sites. The limiting effect of GS on cell size, here is V dia , could be explained by the "selfish DNA hypothesis". The theory holds that the self-replicating genes with no phenotypic expression are favored by natural selection at the genome level (Doolittle & Sapienza, 1980;Orgel & Crick, 1980). In this study, the positive relationship between GS and V dia under the same environmental conditions is consistent with the "selfish DNA hypothesis." In contrast, our results showed that the relationships between GS and V dia became uncertain across different environmental condi- ous studies have reported that great V dia is associated to great K S and low ÀP 50 because of V dia as the anatomic structure of K S and P 50 (Lens et al., 2011;Scoffoni et al., 2016;Tataranni et al., 2015). In the present study, we also found the strong positive relationship between V dia and K S and the strong negative relationship between V dia and ÀP 50 ( Figure 5). K S and P 50 are two key indicators representing plants' hydraulic efficiency and safety, respectively, and they are often at a trade-off (Fan et al., 2017;Grossiord et al., 2020;van der Sande et al., 2019) ( Figure S4). Species that evolved specific K S and P 50 in different climate conditions are thought of as adaption or plasticity (Choat et al., 2018;Liu et al., 2019). In warmer and moister climates, great K S and low ÀP 50 evolved because of sufficient water supply and low hydraulic failure risk. Conversely, low K S and great ÀP 50 evolved in the arid and relatively cold environmental conditions because of the insufficient water supply and high hydraulic failure risk (Choat et al., 2018;Grossiord et al., 2020;Lens et al., 2011) ( Figure S3). The environment selection of K S and ÀP 50 is ultimately reflected by anatomic structure (Pfautsch, 2016;Scoffoni et al., 2016). Thereby, the concerted regulation of environmental factors on V dia could be explained by the environmental selection on hydraulic performance.
The shift of the limiting effect of GS on V dia by environmental pressures indicates the combinative regulation of plant traits by intrinsic and environmental factors. GS is the intrinsic trait regulating cell size where the large GS would contribute to big V dia ; meanwhile, the large GS is also the burden depressing cell fitness and is accompanied by slow metabolism and growth rate (Simonin & Roddy, 2018;Vinogradov, 2003). It is thus possible that seed plants with large GS show less environmental plasticity compared with plants with small GS. Indeed, we found that the relationship between GS and V dia was overall positive in gymnosperms consistent with the theory that GS limits cell size; however, we found that the limiting effect of GS on V dia was completely reversed in angiosperms, regardless of insignificance ( Figure 4a). It implies the great plasticity of angiosperms (small GS) than gymnosperms (large GS), which possibly explains the great success of angiosperms relative to gymnosperms in the terrestrial ecosystem, particularly in wet-hot climates (Lamy et al., 2014). Thereby, the evolution of genome downsizing and the plasticity to changed environment together regulate plant growth, survival, and reproduction through deep time. Combined together, the findings in this study using xylem vessel cell diameter provided the new evidence of the plant performance regulated by the intrinsic and environmental drivers.
In conclusion, we provided clear evidence that the limiting effect of GS on V dia could be shifted by environmental factors in the present study. The regulation of V dia by environmental factors could be explained by the environmental selection on hydraulic performance.
The findings provided critical insight and deepened our understanding of the combinative regulating mechanism on plant performance by intrinsic and environmental factors. The comprehensive cell size driving mechanism would initiate wide researcher interests in the relevant fields and narrow the gap between molecular and macroecology research.

ACKNOWLEDGMENTS
The authors thank the funding by the Key Research Program of Frontier Sciences, CAS (grant no. QYZDJ-SSW-DQC040) and the National Natural Science Foundation of China (grant no. 32171876) and the technical assistance by Liu RH.

CONFLICT OF INTEREST
The authors do not have any conflict of interest to declare.

AUTHOR CONTRIBUTIONS
Wenzhi Zhao and Xiangyan Feng provided the idea and designed the experiment. Xiangyan Feng, Linfei Zhong, Hai Zhou, Jingwen Bi, Huma Batool, and Xintan Zhang conducted the investigation, processed data, and prepared the figures. Xiangyan Feng and Wenzhi Zhao wrote and reviewed the manuscript.